Method for early detection, detection of the severity and for a treatment-accompanying assessment of the course of a sepsis

ABSTRACT

Disclosed is a method for early detection, detection of the severity as well as for a treatment-accompanying assessment of the course of a sepsis as well as means for carrying out said method. According to the invention, the content of the peptide procalcitonin and/or of a partial peptide formed therefrom which is not the mature calcitonin is determined in a sample of a biological liquid of a patient. From the determined presence and amount of the certain peptide, a conclusion is made about the presence of a sepsis, its severity and/or the success of a therapeutic treatment.

The invention relates to a method for sepsis diagnosis, in particular toearly detection and detection of the severity of a sepsis as well as fora treatment-accompanying survey of the therapeutical success of a sepsistreatment which is based on the new knowledge that a certain peptideknown per se and possibly certain of its fragments represent reliablebiological markers for diseases of this kind appearing in highconcentrations and which may be determined relatively simple accordingto classical detection measures.

According to a more modern understanding of this disease, the term"sepsis" as used in the present application summarizes clinicalpictures, for which, as a rule, fever, leucocytosis, consciousnesschanges, a hyperdynamic circulation ("warm shock"), and ahyper-metabolic status, mainly as a consequence of the invasion of thenormally sterile tissue by microorganisms, are observed, whereas thepositive detection of germs in the blood, which was previouslyunderstood to be a characteristic for a sepsis, has become lessimportant for the diagnosis "sepsis". For in clinical studies it couldbe shown that the prognosis of patients with sepsis is not dependent onthe severity of an infection, in particular a bacterial infection, buton the severity of the septic reaction of the organism (see G. Pilz, S.Fateh-Moghadam and K. Werdan in: Krankenpflege-Journal 29 (1991), pp.483-492 and publications cited therein). Accordingly, in addition to thepositive blood culture or instead of it, for a sepsis assessment atpresent various laboratory parameters and hemodynamic parameters aredetermined and taken into account for making a diagnosis and assess thecourse of the disease, if necessary, using computer-aided so-calledScore systems, such as the APACHE (Acute Physiology and Chronic HealthEvaluation) II Score described in the above-indicated publication.However, so far no individual parameter suitable as a reliablebiological marker is known, the determination of which is highlyexpressive for a sepsis diagnosis. All parameters used up to now haveeither an insufficient specificity or do not allow a reliable assessmentof the severity of a sepsis and no therapy survey and, in addition tothis, the determination of substances, such as the tumor necrosis factor(TNF), or interleukines, such as interleukin 6 (IL-6), is much toocomplicated, expensive and/or time-consuming for a bedsidedetermination.

Therefore, there is still an urgent need of a reliable biological markerwhich can be determined relatively easy and the qualitative andparticularly quantitative determination of which is highly indicativefor making a diagnosis and assessing the progression of a sepsis.

It is the object of the present invention to provide a method for earlydetection and for detection of the severity of a sepsis wherein a newbiological marker is determined in a way which is also practicable underclinical conditions, the determination of which gives highly relevantresults for making a diagnosis and assessing the progression of asepsis.

This object has been achieved by a method and means according to theclaims. The invention is based on the surprising finding that thepeptide procalcitonin known per se and, if necessary, certain of itshigher molecular cleavage products, represent highly relevant biologicalmarkers for sepsis and that their concentrations in samples ofbiological liquids of patients allow highly relevant conclusions on theseverity of a septic disease and, thus, represent valuable parametersfor the progression assessment and therapy survey of a sepsis.

Therefore, the possibility of a sepsis diagnosis by determination of thepeptide procalcitonin is of a great practical interest, since otherknown possible biological markers appearing in the case of sepsis, suchas certain cytokines (interleukines, TNF) represent unstable moleculeswhich normally are present only in very small concentrations, so thattheir determination is much too complicated, time-consuming and thusexpensive for a routine bedside diagnosis. As could be determinedaccording to the present invention--completely surprising with respectto the previous medical knowledge--the procalcitonin content isenormously elevated in the case of a sepsis, so that concentrations inthe ng range (above 1 ng, in particular above 10 ng up to 500 ng andmore per ml serum or plasma sample) are obtained, whereas for healthypersons with the known best methods of procalcitonin determination noprocalcitonin content can be detected (concentrations below 0.1 ng/mlsample). At the same time in the case of a sepsis no increasedcalcitonin concentrations are observed according to the invention, whichis remarkable for the reason that up to the present, as a rule,procalcitonin was regarded as calcitonin precursor, the appearance ofwhich also leads to a calcitonin formation.

The peptide procalcitonin to be determined with the method according tothe invention and its possibly appearing proteolytic cleavage productsare known, and determination methods suitable for a quantitative andqualitative immunodiagnostic determination are also known.

Procalcitonin is a peptide of 116 amino acids, and up to the present itwas known about it that it appears as an intermediate of thetranslation/expression of a certain gene (CALC-1), leading to theformation of the peptide hormone calcitonin, in a plurality of tissues,in particular in the thyroid C cells and in tumor tissue, as precursorof calcitonin, with this original gene (CALC-1), apart from theformation of procalcitonin also controlling the formation ofprocalcitonin-gene-related peptide, and distinctly differing from it byits length and in the sequence of the amino acids 51 to 116 of theprocalcitonin (see J. Biol. Chem., 261, 31(1986), pp. 14386-14391).

According to the general knowledge, procalcitonin is a proteolyticaldegradation product of the primary protein preprocalcitonin formed byone certain type of gene expression of the gene CALC-1, and in the knowncases of its appearance, as a rule, undergoes a further stepwisedegradation under release of mature calcitonin which corresponds to asequence of 32 amino acids (amino acids 60 to 81 of the procalcitonin).Among others, in this process at first two larger peptides are formedwhich may be designated N-procalcitonin-(1-57)-peptide, andC-procalcitonin-(60-116)-peptide, with the latter peptide being furthersplittable to the hormones calcitonin and to the peptide known askatacalcine (Biochem. J. 256, (1988) 245-250; and Cancer Research 49(1989), 6845-6851). From J. Biol. Chem. 226, 36, pp. 24627-24631 it hasrecently been known that, apart from the procalcitonin described in theabove publications, also a variant of the procalcitonin is formed inhuman thyroid C cells which differs from the first by the last 8 aminoacids of the C terminus. Also this peptide is to be regarded as"procalcitonin" in the sense of the present invention, since at presentthe immunodiagnostical determination methods used in the development ofthe present invention do not allow to differentiate between the twoprocalcitonins and possible other closely related peptides. Therefore,"procalcitonin" in the sense of the present invention stands for one ora plurality of peptides including the known molecule procalcitonin, theabove-described variant thereof, having an amino acid compositiondeviating therefrom at the C terminus and possible further existingvariants with a comparable reactivity in the selectiveimmunodiagnostical determination methods used for their determination,in particular in the monoclonal immunoradiometric assay described in thefollowing with reference to the publication Cancer Res. 49, (1989),6845-6851.

All these peptides contain peptide sequences of 57 amino acids or more,in particular 116 amino acids like the complete procalcitonin, andcorrespond to the known sequence or represent partial sequences thereof,with deviations in the region of the amino acids which correspond to theamino acids 108 to 116 of the procalcitonin being possible.

Concerning the previous trials to use the detection of calcitonin andrelated peptides for diagnostic purposes, it is known that calcitonin isa valuable biological marker (tumor marker) for numerous malignantdiseases, and a plurality of immunodiagnostical determination methodshas already been developed for the specific determination of calcitoninwhich methods are carried out using specific monoclonal antibodies (see,for example, Clinica Chimica Acta (1988) 174, pp. 35-54; Immunol., Vol.141, pp. 3156-3163; J. Endocr. (1988) 119, pp. 351-357).

Also for determining calcitonin precursors, such as procalcitonin andC-procalcitonin-(60-119)-peptide, an immunodiagnostic determinationmethod was already developed which works according to the principle ofan immunoradiometric assay (IRMA) and which, apart from calcitonin,allows to selectively determine procalcitonin in that a pair ofmonoclonal antibodies is used, one of which is specific for regionsexternal of the calcitonin sequence (the amino acids 1 to 11 of thekatacalcin or the amino acids 96 to 107 of the procalcitonin), and whichis used, for example, in an immobilized form for the extraction ofpeptides from the analysis sample containing this sequence, whereas thesecond marked monoclonal antibody, used for forming the IRMA sandwich,is specific for the region corresponding to the amino acids 11 to 17 ofthe calcitonin (amino acids 70 to 76 of the procalcitonin). In thismanner in the immunoassay only those peptides are detected which havethe calcitonin regions as well as the amino acids 1 to 11 of thekatacalcin region and thus represent either a complete procalcitonin ora peptide with the indicated areas obtained therefrom, such as theC-procalcitonin-(60-116)-peptide (see Cancer Res. 49 (1989), pp.6845-6851). From the plurality of monoclonal antibodies being availablethose have been selected (designations mAbKC01 and mAbCT08) in the knownmethod which had association constants in the range of K_(asn)=0.9-3.0×10¹⁰ M⁻¹. This known method may be used for the determinationmethod according to the invention directly or by using similarmonoclonal antibodies which can be obtained on the basis of thedisclosure in J. Immunol., Vol. 141, No. 9, (1988), pp. 3156-3163, inwhich for a determination defining the procalcitonin increase in aparticularly clear and easily evaluable way for clinical purposes pairsof antibodies should be used which have similar high affinities as thosedescribed above.

For example, pairs of monoclonar antibodies useful for the method of theinvention can be produced by using CT-TT (Calcitonin-Tetanustoxoid) asimmunogen according to previously described immunization procedures(Motte et al., J. Immunol. 138 (1987), 3332) for the production ofantibodies having binding characteristics similar to that of CT21.Antibodies having binding characteristics similar to that of KC01 can beobtained by immunizing Biozzi high responder mice (Biozzi et al., J.Exp. Med. 132 (1970), 152) with KC-TT (katacalcin-tetanustoxoid) wherebyaccording to the procedure described four injections are used consistingof 15 μg of peptides each by different routes; s.c. in Freund's completeadjuvant (FCA), s.c. in Freund's incomplete adjuvant (FIA), i.p. in FIAand i.v. in 0.15 mol/l sodium chloride. The immunization schedules span13 to 30 weeks. 3 days after the last i.v. injection of the conjugatethe mice are sacrificed, the spleen is removed and splenocytes are fusedwith the NS1 mouse myeloma cell-line by using 40% polyethylene glycol(Bellet et al., J. Clin. Endocrinol. Metab. 56 (1983), 530). Thehybridoma supernatants can be screened for specific antibody productionby using an ELISA assay. After cloning by limiting dilution, the hybridcells are implanted i.p. in BALB/c nude (nu/nu) mice and the resultingascites fluids are collected after 10 to 14 days. The monoclonalantibodies can be purified from the ascites fluids using 50% ammoniumsulfate precipitation at 4° C. and protein A chromatography as describedin Manil et al. (J. Immunol. Methods 90 (1986), 25). The haptene-carrierconjugates CT-TT or KC-TT can be prepared by separately linking CT or KCto TT by using glutaraldehyde as a coupling agent (Audibert et al.,Proc. Natl. Acad. Sci. U.S.A. 79 (1982), 5042).

For the determinations described in the following examples, a pair ofmonoclonal antibodies has been used which included the above-indicatedantibody mAbKC01 and an antibody mAbCT21, with the antibody mAbCT21regarding its binding to the procalcitonin molecule as well as regardingits affinity being very similar to the antibody mAbCT08 described in theabove publication (association constant K_(asn) =3.0×10¹⁰ M⁻¹).Hybridomas producing the monoclonal antibodies KC01 and CT21 weredeposited at "DSM--Deutsche Sammlung yon Mikroorganismen andZellkulturen GmbH", Mascheroderweg 1B, 38124 Braunschweig, Germany,according to the provisions of the Budapest Treaty under deposit numbersDSM ACC2124 and DSM ACC2125, respectively, on Apr. 20, 1993.

The described method according to Cancer Res. 49, (1989), pp. 6845-6851,provides concentration values of the content of a procalcitonin or theC-procalcitonin-(60-119)-peptide or of both of them in samples or, ifthe stabilities of both peptides are comparable, a value for the initialtotal concentration of the procalcitonin in the sample. With respect tomethods which may be used for determining procalcitonin in accordancewith the present invention, we explicitly refer to the above-mentionedpublication and the publications cited therein, the contents of whichare included in the present application by reference for supplementingthe disclosure of the present application.

In the above-indicated publication, the attempt was made to carry outthe determination of procalcitonin in order to check its suitability astumor marker. It has been established that the procalcitonin andcalcitonin levels in tumor patients were of parallel behaviour fromwhich the conclusion was drawn that both were derived from neoplastic Ccells from the thyroid. In the above-mentioned publication it wasfurther established that the procalcitonin levels were also increased inpatients who did not suffer from malignant diseases, but from certainserious virus infections. In these cases, the calcitonin levels were notsimultaneously elevated. These patients were not septic patients andtheir diseases did not have any relation to sepsis diseases.

After it was surprisingly established according to the present inventionthat there is a close correlation between the procalcitonin levels andthe presence and severity of a sepsis, additional considerations weremade about the cause of the increase of the procalcitonin level withouta simultaneous increase of the calcitonin level in the case of sepsis aswell as--to a clearly smaller extent which allows it in most cases todirectly distinguish these cases over sepsis--in patients suffering fromcertain serious virus diseases. Since in one patient with sepsis whounderwent total thyroidectomy, nevertheless the increase of theprocalcitonin to a level significant for a sepsis could be detected, itwas clear that in the case of sepsis the procalcitonin is not formed inthe thyroid, but that another organ is competent for it. If with respectto the increased procalcitonin level in the case of virus hepatitis as aworking hypothesis it is assumed that this other organ is the liver, theincrease of the procalcitonin level could be explained in the first caseas a direct effect of the virus disease on the hepatocytes and in theother case as an indirect, but more effective influence of theendotoxines produced by the bacteria responsible for the sepsis on thesame hepatocytes. However, it has to be underlined that this ex-postexplanation represents a working hypothesis and not a theory proven byexperiments.

The appearance of increased procalcitonin levels in the case of seriousvirus diseases has the effect for the method for sepsis diagnosisaccording to the invention that, if the procalcitonin levels areelevated only to a relatively small extent up to such values which canalso be found in cases of serious virus diseases, the presence of such avirus disease must be excluded before a sepsis diagnosis is made.

Further, in patients with chronic renal failure and therefore disorderedpeptide excretion it should perhaps be expected that the levels ofpeptides like procatcitonin are elevated, but that this elevation doesnot have the same clinical relevance as it is the case in patients whichare healthy in this respect. However, the physician establishing theclinical diagnosis can easily take into account these circumstances.

The present invention is not restricted to a use of the above-describedknown special determination method for the determination ofprocalcitonin, but includes also other determination methods known perse, to which belong also those methods using other monoclonal orpolyclonal antibodies, for example, those methods working with aspecificity for the N-procalcitonin-(1-57)-peptide and, in particular,its amino acids 51 to 57. Thus, it could be shown for a common use ofpolyclonal antibodies against regions of theN-procalcitonin-(1-57)-peptide instead of mAbKC01 together with themarked monoclonal antibody binding to the calcitonin region of theprocalcitonin used in the described method that in both cases analogousconcentration values were obtained for the procalcitonin content which,on the basis of the fact that, if the indicated polyclonal antibodiesare used, the detected regions are present within one molecule only inthe case of the intact procalcitonin peptide, suggests the conclusionthat in the case of a sepsis the levels of the intact procalcitonin arein fact elevated and the partial peptides formed thereof are ofsecondary importance at the most.

In principle, the method according to the invention may be carried outalso by determining the procalcitonin in a way other thanimmunodiagnostic, for example, by means of HPLC, if such methodsproviding sufficient sensitivity and specificity exist or can bedeveloped.

Although moreover the determination of the procalcitonin according tothe invention is carried out at present mainly in serum or plasmasamples, the method according to the invention in principle includesalso determinations of procalcitonin in other biological fluids, such aswhole blood and urine, if it should turn out that also in these fluidsprocalcitonin levels can be measured in a reproducible manner.

Concerning the state of the art it is additionally indicated that inSurgery, Vol. 108, 6 (1990), pp 1097-1101, it is reported that inpatients with sepsis the plasma level of the peptide CGRP, which isrelated to calcitonin, was slightly elevated in the pg range (14.9+3.2pg/ml compared to 2.0+0.3 pg/ml in control persons). These findings donot allow to draw any conclusion about the levels of other, relatedpeptides, and the significantly lower absolute concentrations andsignificantly lower relative increases in the case of sepsis incomparison with the normal concentration compared to the increase in themethod according to the invention suggest that the determination of CGRPas sepsis marker is not suitable.

Further, in Lancet 1, (1983), p. 294 it has been reported that in thecase of serious meningococcaemia in children the observed calcitoninlevels were raised two times to three times, however, in a followingpublication in Pediatr. Res. 18, (1984), p. 811 it was corrected thatthe determined substance probably is not the intact calcitonin, but noindication was made what substance was actually measured. The observedincrease to approximately three times the normal value in the describedcases has to be compared with an increase of procalcitonin in the methodaccording to the invention in the range of a 1000-fold increase, whichshows that the indicated publications do not represent a disclosurerelevant for the present invention.

The method according to the invention will now be described in moredetail with reference to clinical data producing evidence for therelevance of the delivered information.

All procalcitonin determinations have been carried out according to themethod of determining procalcitonin described in Cancer Res. 49, (1989),pp 6845-6851, using the monoclonal antibodies KC01 (DSM ACC2124) andCT21 (DSM ACC2125) (see above).

The examples illustrate the invention but should not be construed aslimiting the invention.

EXAMPLE 1 Determination of the procalcitonin levels in childrenhospitalized for various diseases

The procalcitonin levels (pCT) of different groups of childrenhospitalized for various diseases have been determined.

The results are summarized in Table 1.

It may be seen that in sepsis patients up to 180 ng/ml pCT(procalcitonin level) were obtained, whereas the pCT values for "normal"virus diseases maximally increased to 2 ng/ml, only for extremelyserious virus diseases of intestine and liver the values raised to 16ng/ml and in one case to 35 ng/ml

                  TABLE 1                                                         ______________________________________                                        Serum levels of pCT in children with bacterial and                            viral infections                                                                               pCT                                                          Group   Age (yr) (ng/ml)   Clinical Details                                   ______________________________________                                        Controls                                                                              0.3-10   <0.1      Children hospitalized for                          (n = 20)                   various diseases with no                                                      infections                                         Bacterial                                                                             0.5-8.5   16-180   5 with meningitis (3                               infection                  hemophilus, 2 meningo-                             children                   coccus), 1 with pulmonary                          (n = 7)                    pneumococcosis, 1 with                                                        staphylococcia and                                                            Steven-Johnson's syndrome                          Newborn NN        13-160   6 newborn with positive                            (n = 6)                    blood culture (Escherichia                                                    Coli, Streptococcus B,                                                        enterobacter, listeria)                            Viral                                                                         infection                                                                     (n-10)  NN-9     <0.1-2.0  3 with Lymphocytic                                                            Meningitis 7 with various                                                     viral infections                                                              (elevated interferon)                              (n = 3) 0.1-5    1.1-16    2 with rotavirus                                                              1 with coronavirus infections                      (n = 3) 2-5      1.5-35    All with hepatitis A                               Congenital                                                                            NN       <0.1      All with subclinical disease                       toxoplas-                                                                     mosis                                                                         (n = 6)                                                                       ______________________________________                                    

EXAMPLE 2

Correlation of the pCT levels in patients with sepsis, for whom thecourse of the disease has simultaneously been observed according to theAPACHE II Score, with the severity of their illness

In20 septic patients who have been treated by an i.v. Pseudomonas-IgGsepsis therapy after cardiac operations, the pCT levels have beenobserved for five days with their illness state simultaneously beingevaluated according to the APACHE II Score. The results are summarizedin the following Table 2.

                                      TABLE 2                                     __________________________________________________________________________     ##STR1##                                                                     __________________________________________________________________________     x ± SEM                                                                    1p < 0.05 (MW)                                                                2p < 0.05 (Wilcoxon)                                                          3p < 0.05 (Chi.sup.2)                                                    

From Table 2, it may be clearly taken that in the case of response to asuccessful sepsis therapy (Responder) the pCT levels decreased with theimprovement of the clinical status, whereas they remained nearlyunchanged high in the case of non-response (Non-Responder) to thetreatment. It may also be seen that in the case of non-responders, theinitial pCT levels were significantly higher than those of theresponders and thus, the illness severity of the former was greater. Ascompared with the values of the APACHE II Score, the pCT valuessignificantly clearly represent the different severity grades of thesepsis, which is also confirmed by the lethality figures.

These results show further that the accompanying determination of pCTlevel during a sepsis treatment at an early state gives reliableinformation on the treatment success, so that if necessary, an earlydecision in respect of a change of the selected treatment, for example,the selection of another antibiotic preparation is possible.

Similar results could also be derived in the case of burn patients, inwhom a sepsis developed in connection with skin transplantations, whichwere treated with various antibiotic preparations. A treatment successwas always accompanied by a significant decrease of the pCTconcentration, and in one case it was possible to early correctnon-response to a first treatment with a first antibioticpreparation--which could be detected since the pCT concentrationsremained constant--by changing the antibiotic preparation with theresponse to the second antibiotic preparation being recognizable in thatthe pCT level dropped immediately.

I claim:
 1. Method for early detection of a sepsis in a patient in needthereof comprising: determining in a sample of a biological liquid of apatient the concentration of procalcitonin, the determined presence andamount of procalcitonin being indicative of the presence of a sepsis. 2.The method according to claim 1, characterized in that procalcitonin andN-procalcitonin (amino acids 1-57)-peptide and C-procalcitonin-(aminoacids 60-116)-peptides are determined.
 3. Method according to claim 1 or2, characterized in that the corresponding peptide or peptides aredetermined in an immunodiagnostic method by use of a monoclonal antibodyor a combination of a first monoclonal or polyclonal antibody with asecond monoclonal antibody, which as such or combined with each otherhave a specificity for procalcitonin or peptides formed therefrom, andwhich allow to differentiate between them and mature calcitonin and theCGRP peptide.
 4. Method according to claim 3, characterized in thatprocalcitonin is determined by means of an immunometric assay whereinfor binding the procalcitonin from the sample a first monoclonalantibody is used binding the procalcitonin in another region as thesecond monoclonal antibody used for marking, so that a differentiationbetween procalcitonin and its proteolyic degradation products includingcalcitonin is possible.
 5. Method according to claim 4, characterized inthat, apart from the first monoclonal antibody, for bindingprocalcitonin at least one further monoclonal antibody is used whichbinds the procalcitonin molecule in another region.
 6. Method accordingto claims 1, characterized in that procalcitonin is determined by meansof an immunometric assay which selectively detects the completeprocalcitonin and C-procalcitonin-(60-116)-peptide, if present. 7.Method according to claim 1, characterized in that procalcitonin isdetermined by means of an immunometric assay which selectively detectsthe complete procalcitonin and N-procalcitonin-(1-57)-peptide.
 8. Methodaccording to claim 6, characterized in that procalcitonin contents ofmore than 1 ng/ml sample are indicative of the possible .presence of asepsis and that procalcitonin contents in the range of 10 ng/ml to 500ng/ml and higher correlate with an increasing severity of the sepsis anda worsened prognosis.
 9. Method according to claim 8, characterized inthat simultaneously in a parallel determination the calcitonin level isdetermined and that the absence of evidence for elevated calcitoninlevels allows the sepsis diagnosis to be made.
 10. Method according toclaim 8 or 9, characterized in that procalcitonin levels in the range ofapproximately up to 20 ng/ml allow the sepsis diagnosis to be made, ifthe presence of a serious virus disease as a reason for the elevatedprocalcitonin level can be excluded.
 11. Method according to claim 1,characterized in that a serum or plasma sample is used as the sample ofa biological liquid.
 12. Method according to claim 1, characterized inthat the monoclonal antibody is the monoclonal antibody KC01 (DSMACC2124) or the antibody CT21 (DSM ACC2125).
 13. The method of claim 1wherein the amount of the peptide detected indicates the severity of thesepsis or the success of a therapeutic treatment.
 14. The method ofclaim 13 wherein the antibody is selected from the group consisting ofmonoclonal antibodies KC01 and CT21.
 15. The method of claim 1, whereinthe concentration of procalcitonin is determined immunologically.